Regulation of human immunodeficiency virus type 1 envelope glycoprotein fusion by a membrane-interactive domain in the gp41 cytoplasmic tail

Abstract

Truncation of the human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) gp41 cytoplasmic tail (CT) can modulate the fusogenicity of the envelope glycoprotein (Env) on infected cells and virions. However, the CT domains involved and the underlying mechanism responsible for this "inside-out" regulation of Env function are unknown. HIV and SIV CTs are remarkably long and contain amphipathic alpha-helical domains (LLP1, LLP2, and LLP3) that likely interact with cellular membranes. Using a cell-cell fusion assay and a panel of HIV Envs with stop codons at various positions in the CT, we show that truncations of gp41 proximal to the most N-terminal alpha helix, LLP2, increase fusion efficiency and expose CD4-induced epitopes in the Env ectodomain. These effects were not seen with a truncation distal to this domain and before LLP1. Using a dye transfer assay to quantitate fusion kinetics, we found that these truncations produced a two- to fourfold increase in the rate of fusion. These results were observed for X4-, R5-, and dual-tropic Envs on CXCR4- and CCR5-expressing target cells and could not be explained by differences in Env surface expression. These findings suggest that distal to the membrane-spanning domain, an interaction of the gp41 LLP2 domain with the cell membrane restricts Env fusogenicity during Env processing. As with murine leukemia viruses, where cleavage of a membrane-interactive R peptide at the C terminus is required for Env to become fusogenic, this restriction of Env function may serve to protect virus-producing cells from the membrane-disruptive effects of the Env ectodomain.

FIG. 1.

Mutagenesis of the HIV-1 gp41 cytoplasmic domain. Sequence alignments of gp41 cytoplasmic tails are shown for HXBc2, JRFL, and 89.6, using the numbering for HXBc2. Highlighted domains include the C-terminal region of the membrane-spanning domain (msd) and the amphipathic alpha-helical domains LLP-1, LLP-2, and LLP-3 (18, 43, 45, 58, 59, 87). A largely conserved palmitoylated Cys is shown (solid arrow) prior to the beginning of LLP-2, as is the position of a frameshift mutation (gray arrow) described for the CD4-independent variant of HXBc2, 8x (49). Mutations introduced into HXBc2 are shown, including stop codons (asterisks) at the indicated positions, i.e., amino acids 733 (Ile), 753 (Leu), 764 (Cys), 771 (Leu), and 808 (Lys), and the 8x fs mutation at position 706. An additional mutant (HXB-C764A/771*) is shown containing a stop codon and a Cys-to-Ala mutation at position 764 and a stop codon at position 771. For JRFL and 89.6, the mutations introduced included the 8x fs mutation and stop codons introduced to produce tails comparable in length to those with the fs mutations.

FIG. 2.

Increased fusogenicity with truncations in the gp41 cytoplasmic tail. The fusion activity of HIV-1 Envs was assessed in cell-cell fusion assays with target cells expressing CD4 and CXCR4, as described in Materials and Methods. Envs included those of parental HXBc2 (HXB), 8x (a CD4-independent variant of HXBc2), and HXB-fs (HXBc2 containing a frameshift mutation at position 706 in the cytoplasmic tail) and HXBc2-derived Envs containing stop codons in their cytoplasmic tails at the indicated amino acid positions (see Fig. 1). The results of luciferase activity measurements were normalized to the level of fusion obtained with parental HXB. Envs with cytoplasmic tails terminating at amino acid 764 or before exhibited greater fusion activities, whereas Envs with stop codons at positions 771 and 808 had activities similar to that of HXB. The data were compiled from three experiments, with each done in triplicate. Standard errors of the means are shown.

FIG. 3.

Exposure of CD4-induced epitopes after gp41 cytoplasmic tail truncations. The binding of antibody 17b, which reacts with a CD4-induced epitope on gp120, was assessed on Envs expressed in QT6 cells by using a previously described binding assay (28, 29). The results were normalized to the amount of binding seen on the CD4-independent 8x Env, which has been shown to expose this epitope in the absence of CD4 binding (38, 49). Results are shown for the panel of HXB-derived Envs evaluated in Fig. 1. The data are from three experiments, with each done in triplicate. Standard errors of the means are shown.

FIG. 4.

Fusion kinetics of HXBc2-derived Envs with gp41 cytoplasmic tail truncations on SupT1 cells. The panel of HXBc2-derived Envs shown in Fig. 1 were expressed in HeLa cells, and the rate of fusion was assessed on SupT1 cells (CD4⁺ and CXCR4⁺) labeled with calcein AM (see Materials and Methods). An additional Env is shown containing a stop codon at position 771 in combination with a Cys-to-Ala mutation at position 764, which ablated a palmitoylation site (Fig. 1). Lines represent fits to the sigmoidal equation f = a/{1 − exp[−b(t − t_1/2)]} (36). Values for half-maximal fusion times are shown in Table 1. Envs with cytoplasmic tails truncated at or before position 771 exhibited accelerated fusion kinetics compared to Env 808* and wild-type HXBc2.

FIG. 5.

Fusion kinetics of R5- and dual-tropic Envs with prematurely truncated cytoplasmic tails. (A) Fusion kinetics of JRFL and 89.6 Envs with a CT frameshift mutation at a position analogous to that in 8x or a premature stop codon at position 748 were assessed on CEM/CCR5 cells as described in Materials and Methods. Times to half-maximal fusion are shown in Table 1. (B) The dual-tropic 89.6 Envs were also evaluated with the CD4⁺ CXCR4⁺ cell line SupT1. Although the differences were less impressive, 89.6-fs and -stop Envs each exhibited faster kinetics than the 89.6 wild type (Table 1).

FIG. 6.

Surface expression of Env constructs. HXBc2-derived Envs containing a stop codon at position 733 or the 8x frameshift mutation were expressed in HeLa cells, and surface expression was evaluated 24 h later by fluorescence imaging microscopy (see Materials and Methods). Controls included parental HXBc2 and the CD4-independent 8x Env. The negative controls were HeLa cells infected with vTF or transfected with a PSP plasmid. No differences in Env expression were seen for these or the other plasmid shown in Fig. 1.

FIG. 7.

Sensitivity of cytoplasmic tail mutant Envs to enfuvirtide inhibition. The sensitivity of fusion to inhibition by enfuvirtide is shown for the HXBc2-derived Envs HXB-fs and 733* compared to the parental HXBc2 and CD4-independent 8x Envs. For each Env, the % fusion on SupT1 cells in the absence of inhibitor is shown, and data were plotted using Sigma Plot. IC₅₀s are shown for each Env.

FIG. 8.

Effects of cytoplasmic tail on CD4 independence. Cell-cell fusion assays were performed with QT6 cells transfected with control pCDNA3 or with CD4, CXCR4, or CD4 in combination with CXCR4. The Envs used were those of HXB, 8x, and HXB-fs and an 8x Env containing a full-length cytoplasmic tail (8x/HXB-TM). The parental HXB and 8x Envs were CD4 dependent and independent, respectively, as expected, and HXB-fs, while exhibiting enhanced fusion, remained CD4 dependent (28, 29). However, restoring a full-length CT to 8x considerably reduced both the fusion efficiency and CD4 independence.